In the commercial production of crops, it is desirable to easily and quickly eliminate unwanted plants (i.e., “weeds”) from a field of crop plants. An ideal treatment would be one which could be applied to an entire field but which would eliminate only the unwanted plants while leaving the crop plants unharmed. One such treatment system would involve the use of crop plants which are tolerant to a herbicide so that when the herbicide was sprayed on a field of herbicide-tolerant crop plants, the crop plants would continue to thrive while non-herbicide-tolerant weeds were killed or severely damaged. Ideally, such treatment systems would take advantage of varying herbicide properties so that weed control could provide the best possible combination of flexibility and economy. For example, individual herbicides have different longevities in the field, and some herbicides persist and are effective for a relatively long time after they are applied to a field while other herbicides are quickly broken down into other and/or non-active compounds. An ideal treatment system would allow the use of different herbicides so that growers could tailor the choice of herbicides for a particular situation.
Crop tolerance to specific herbicides can be conferred by engineering genes into crops which encode appropriate herbicide metabolizing enzymes and/or insensitive herbicide targets. In some cases these enzymes, and the nucleic acids that encode them, originate in a plant. In other cases, they are derived from other organisms, such as microbes. See, e.g., Padgette et al. (1996) “New weed control opportunities: Development of soybeans with a Roundup Ready® gene” and Vasil (1996) “Phosphinothricin-resistant crops,” both in Herbicide-Resistant Crops, ed. Duke (CRC Press, Boca Raton, Fla.) pp. 54-84 and pp. 85-91. Indeed, transgenic plants have been engineered to express a variety of herbicide tolerance genes from a variety of organisms.
For nearly two decades, corn, soybean, and cotton farmers have relied on glyphosate and glyphosate resistant crops for weed control. While favored for its efficacy, economy and convenience, the onset of glyphosate-resistant weeds, now numbering 26 species (Heap, 2014), signals a need for new herbicide and trait systems that meet similar criteria (Green and Castle, 2010; Duke, 2012). The silver bullet solution would be another herbicide and trait combination as effective as glyphosate. Unfortunately, prospects for novel herbicide chemistries are not encouraging (Duke, 2012).
Inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD) disrupt production of tocopherols (antioxidants) and plastoquinone (essential for photosynthetic electron transfer) by blocking conversion of tyrosine, through 4-hydroxyphenyl pyruvate (HPP), to homogentisate (Moran, 2014). The result is that the plant cannot protect itself from the radicals generated by light activation of chlorophyll, causing bleaching, necrosis, and death. Registered HPPD inhibitors include mesotrione, tembotrione, sulcotrione, isoxaflutole, and topramezone. HPPD inhibitors are most effective on broad-leaf weeds but control some grasses as well. Currently, HPPD herbicides are selective for use in corn, while soybeans and other dicot crop species are sensitive. A broad spectrum HPPD tolerance trait in soybeans, used in combination with glyphosate tolerance and other traits or selective herbicides, will prolong the positive impact of the glyphosate systems and slow appearance of resistant weeds. Two such products, one for tolerance to isoxaflutole (APHIS, 2009) and the other to mesotrione and isoxaflutole (APHIS, 2012) are in the USDA regulatory approval process. The former uses a Pseudomonas fluorescens HPPD with a single amino acid change (Matringe et al., 2005), while the latter uses HPPD from oat, with a single amino acid change (Hawkes et al., 2010). There are presently no reports of tembotrione or broad spectrum tolerance.
To develop a robust and stable HPPD tolerance trait in soybean, expression patterns of the native gene and localization of native and transgenic protein were studied, and the efficacy of low to moderate expression of a desensitized protein were evaluated. Data regarding the subcellular location of HPPD are ambiguous, perhaps resulting from species diversity. Early work with organelle fractions attributed most HPPD activity to the chloroplast in spinach (Fiedler et al., 1982) or Lemna gibba (Loeffelhardt and Kindl, 1979). Organelle targeting can be conjectured from the observation that the N-terminal sequence of mature HPPD isolated from maize leaf begins at either ala-17 (Fritze et al., 2004) or ala-23 (Yang et al., 2004) with respect to the translated full-length gene. Two HPPD genes identified from EST libraries prepared from cotton tissue were 98.6 percent identical, each with a 23-amino acid sequence deemed likely to function in chloroplast targeting by analysis with the ChloroP prediction program (Moshiri et al., 2007). In the same publication, tomato, but not Brassica HPPD was predicted to have a CTP. Subcellular fractionation supported a cytosolic location of the carrot cell enzyme (Garcia et al., 1997). Although the N-terminal sequence of the purified enzyme was truncated with respect to the translated cDNA, this was attributed to proteolysis during purification. Later, the same authors determined that after PSORT analysis failed to identify a targeting signal within the Arabidopsis HPPD amino acid sequence, native Arabidopsis HPPD heterologously expressed in tobacco was located exclusively in the cytosol (Garcia et al., 1999).
While a number of HPPD crop plants are presently commercially available, improvements in every aspect of crop production, weed control options, extension of residual weed control, and improvement in crop yield are continuously in demand. Particularly, due to local and regional variation in dominant weed species as well as preferred crop species, a continuing need exists for customized systems of crop protection and weed management which can be adapted to the needs of a particular region, geography, and/or locality. A continuing need therefore exists for compositions and methods of crop protection and weed management.